1. Field of the Invention
The present invention relates to a protein biosensor having an increased signal intensity to the concentration of sugars and the use thereof, and more particularly to a fluorescent indicator protein in which fluorescent proteins of different emission wavelength ranges are fused to both ends of a binding protein undergoing a conformational change due to binding of sugars, such that a change in the concentration of various sugars involved in intracellular metabolism can be detected using a change in emission intensities caused by FRET (fluorescence resonance energy transfer), as well as a method for detecting the concentration of sugars using said fluorescent indicator protein.
2. Background of the Related Art
Fluorescent indicator proteins that use the fluorescence resonance energy transfer (FRET) between an enhanced blue fluorescent protein (EBFP) and an enhanced green fluorescent protein (EGFP) or enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), which are genetic mutants of a wild-type green fluorescent protein (wtGFP) derived from jellyfish, initiated for measuring intracellular Ca2+ concentrations with a fusion protein, named ‘cameleon’, consisting of EBFP and EGFP fused to calmodulin and M13 peptide interacting therewith, by R Y Tsien and his colleagues at the University of California, San Diego, in the year 1997 (Miyawaki, A. et al.,Nature, 388:882, 1997; WO 98/40477). Also, in the year 1997, Hellinga and his colleagues constructed a biosensor having excellent sensitivity in vitro by attaching a fluorescent dye to a maltose binding protein (MBP) which is one of periplasmic binding proteins (PBPs) of microorganisms undergoing a conformational change due to binding of ligands, and examples of application to a variety of PBPs have been recently reported (Marvin, J. S. et al., PNAS, 94:4366, 1997; Robert, M. et al., Protein Science, 11:2655, 2002).
On the basis of such studies, in the year 2002, Frommer and coworkers reported results obtained by quantitatively measuring the concentration of maltose in living yeast using a method comprising fusing ECFP and EYFP to both ends of MBP, stably expressing the proteins in living yeast and determining the change in the ratio of the emission intensity of EYFP and that of ECFP (FRET change) (Fehr, M. et al., PNAS, 99:9846, 2002; WO 03/025220). They also reported study results obtained by quantitatively analyzing each of substances in living cells using PBPs such as a ribose-binding protein (RBP), a glucose/galactose-binding protein (GGBP) and a glutamin-binding protein (GlnBP) (Lager, I. et al., FEBS Lett., 553:85, 2003; Fehr, M. et al., JBC., 278:19127, 2003; Okumoto, S. et al., PNAS, 102:8740, 2004). The ‘cameleon’ and its improved derivatives were applied to the monitoring of various substance concentrations in living cells. For example, they were applied to real-time observation of the concentration change of GTP/GDP which play an important role in intracellular signaling (Mochizuki, N. et al., Nature, 411:1065, 2001); measurement of neurotoxin activity of C. botulinum (Dong, M. et al., PNAS, 101:14701, 2004); and quantitative measurement of inositol 1,4,5-trisphosphate in living cells (Tanimura, A. et al., JBC., 279:38095, 2004).
In relation to biosensors for environmental monitoring, new biosensors were developed by reconstructing RBP into a protein capable of binding to environmentally harmful substances, such as TNT, using a genetic manipulation method based on in silico protein design (Looger, L. L. et al., Nature, 423:185, 2003). Also, there was an successful example of reconstructing a novel binding protein in which sucrose can bind to the maltose binding site, which was based on a conformational modification of MBP by inserting β-lactamase into a specific site of MBP with a conformational change due to binding of maltose (Guntas, G. et al., PNAS, 102:11224, 2005). This can be considered as a basic technology which allows the development of a ‘designable FRET sensor’ having a novel binding protein introduced into a binding protein similar to said ‘cameleon’, and thus is useful for the analysis of various cellular functions.
However, because said fluorescent indicator proteins developed in the initial stage have a disadvantage of very weak sensitivity, there is a need to develop a fluorescent indicator protein having excellent sensitivity, which can be used as a means for more precise and quantitative measurement (Fehr, M. et al., Current Opinion in Plant Biology, 7:345, 2004). Also, there were recently attempts to overcome this disadvantage, and in one of such attempts, Miyawaki and his research team have continuously presented study results suggesting that poor signal intensity of fluorescent indicator protein can be increased by circularly permuting the EYFP gene of ‘cameleon’ (Nagai, T. et al., PNAS, 98:3197, 2001; Nagai, T. et al., PNAS, 101:10554, 2004; WO 05/036178). Also, Frommer and coworkers presented study results suggesting that the FRET signal change of each of fluorescent indicator proteins can be greatly increased using an in-frame fusion of inserting a fluorescent protein into genes such as GGBP or GlnBP (Deuschle, K. et al., Protein Science, 14:2304, 2005). However, in order to apply said methods to increase the signal intensity of fluorescent indicator proteins, there are problems in that a high degree of technology is required for genetic manipulation and all development processes should be redesigned at the starting with the initial stage for the construction of novel fluorescent indicator proteins consisting of binding proteins and fluorescent proteins which have different conformational and kinetic characteristics.
Accordingly, the present inventors have made extensive efforts to solve the above-described problems and, as a result, found that the construction of a fluorescent indicator protein having an increased signal intensity to various sugar concentration ranges is possible using a method of inserting a short random peptide between a binding protein and a fluorescent protein or genetically mutating a specific site of a binding protein, to which a substance is to be bound, and then subjecting the resulting fluorescent indicator proteins to high-throughput screening, thereby completing the present invention.